Sandbox wabash 04 fumarase

Fumarase is an enzyme that catalyzes the reaction of malate to fumarate, and of fumarate to malate. The fumarase enzyme being observed here is fumarase C, from e. coli. There are two possible active sites that are found in fumarase, both containing carboxylic acid binding sites. These sites are known as the A site and B site. The key residue in both sites is a histidine residue, which can interact with water and can participate in base catalysis. To determine the actual active site of fumarase, Weaver^[1]. changed the important histidine residue in each possible active site to an asparagine. The experiment measured the specific activity, average activity, and average protein concentration of fumarase upon altering each possible active site individually. The results of the experiment for the mutated fumarase enzymes were compared to that of a wild type enzyme, and the data showed the H129N mutant had similar specific activities to the wild type, but the H188 mutant had a significantly lower specific activity than H129N or wild type. These results show that the active site is the A site because a mutation of H188 yielded significantly reduced enzymatic activity due to the inability of the N188 residue to protonate water and stabilize the highly negative reaction intermediate. When the the substrate (L-malate) is forced to bind to the B site. The results of the experiment show that when this binding to the B site occurs, the efficiency of fumarase is significantly decreased. This The mutation of H129N showed small differences in enzymatic activity compared to the wild type fumarase, which further suggests that the B site is not the true active site. ^[2].

Stability

The true structure of the active site of fumarase (Site A) is formed by residues of histidine (H188), threonine (T100b), serine (98b), asparagine (N141), glutamic acid (E331c), and lysine (K324c), as well as a citrate and a water molecule. The significant amount of Hydrogen bonding that occurs between these residues and the water molecule (W26) stabilizes the active site of the enzyme. Upon substrate binding, the residues shift without losing their hydrogen bonding interactions, which allows for a very stable intermediate. The intermediate contains two negative charges which are stabilized and oriented properly by hydrogen bonds between oxygen and hydrogen atoms in the active site. The water molecule is the key to the catalytic function of fumarase because it helps stabilize the double negative charge of the intermediate by acting as a catalytic acid and accepting a proton from H188 (site).

Disease

Relevance

Structural highlights

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References

1. ↑ Weaver T, Lees M, Banaszak L. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site. Protein Sci. 1997 Apr;6(4):834-42. PMID:9098893
2. ↑ Weaver T, Lees M, Banaszak L. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site. Protein Sci. 1997 Apr;6(4):834-42. PMID:9098893

^[1].